1,3-1,4-β-D-glucanase (lichenase, EC 3.2.1.73) can specifically hydrolyze 1,4-β-D-glucosidic bonds adjacent to 1,3-β-linkages in lichenan or β-D-glucans. The major products of the hydrolysis reaction including cellotriose, cellotetraose and cellopentaose are important nutrients for animals. The enzyme draws much attention because of its broad spectrum of industrial applications. Supplementation of 1,3-1,4-β-D-glucanase in nonruminal animal feed largely increases the feed conversion efficiency and growth rate. In addition to animal feed, 1,3-1,4-β-D-glucanase is also used in beer industry as the enzyme can degrade the high molecular mass β-glucans to prevent reduced yields of extracts, inefficient wort separation and beer filtration. The pelleting and expansion processes in feed industry are performed at 65 to 90° C., and the optimal temperature for the malting processes in beer manufacture is between 50 and 70° C. Therefore, to increase thermostability of 1,3-1,4-β-D-glucanase is highly demanded for various industrial applications.
There are two approaches to obtain a highly thermostable enzyme. The first is to directly clone the enzyme-coding genes from hyperthermophiles and to express the proteins in industrial strains. For instance, Thermotoga maritima cellulase 12A (TmCell2A) that belongs to the GH12 family of glycoside hydrolases shows the strongest activity at 95° C. and has a pH optimum of 5. These characteristics make the enzyme highly valuable in various utilizations, since industrial processes such as plant waste treatments usually involve high temperature and low pH. Nevertheless, the hyperthermophile-derived enzymes usually exhibit low activities in physiological conditions which are between 20 and 37° C., and thus severely limit their applications in aquatic and nonruminal animals. These hyperthermophilic enzymes still need to be modified to meet the requirement for different industrial usages. The second approach to obtain a thermostable enzyme is to directly modify a less thermostable enzyme by genetic manipulations.
Fibrobacter succinogenes 1,3-1,4-β-D-glucanase is classified as a member of the family 16 glycosyl hydrolases and is the only naturally occurring circularly permuted β-glucanase, among bacterial glucanases with reverse protein domains. The C-terminal truncated F. succinogenes 1,3-1,4-β-D-glucanase (TF-glucanase; residues 1-248) exhibits a higher thermostability and enzymatic activity than the full-length enzyme. The structures of TF-glucanase apo-form and in complex with β-1,3-1,4-Cellotriose (CLTR) have been solved. TF-glucanase consists mainly of two 8-stranded anti-parallel β-sheets that are arranged in a jellyroll β-sandwich structure. Residues E11, N44, E47, E56, E60, R137, N139, W141 and T204 are involved in a hydrogen bond network, and residues F40, Y42, W203 and F205 are involved in the stacking interaction between CLTR and TF-glucanase (−3, −2 and −1 subsites). This enzyme has also been well studied by mutagenesis and functional analyses. More importantly, the amounts of secreted TF-glucanase from Pichia pastoris fermentation was approaching 3 g/l by optimizing the codon usage, making the protein production meet the level of industrial manufacturing (range from 1 to 10 g/l). Accordingly, TF-glucanase is an excellent target for directed mutagenesis to be modified as a better product for industrial usage.
Therefore, the present invention directly mutated the TF-glucanase gene in attempt to improve the enzyme activity and thermostability.